SummaryThe bovine leukemia virus (BLV) has a single-stranded RNA genome; it belongs to the Retroviridae family and has eight different genotypes. B lymphocytes are the target cell of BLV, which has a negative impact on the immune system of cattle, making them more susceptible to other diseases of infectious origin; infected dairy cows also have lower production over herd (2.5 to 5 %). The disease is transmitted through consumption of milk and mostly iatrogenic. The aim of this study was to make the diagnosis of BLV by nested PCR technique in 500 samples of blood from Holstein cows, which belonging to several herds located in municipalities with dairyness in the department of Antioquia, it were taken during the months of february to june 2013. It was performed a nested PCR to detect a region of the provirus detecting the viral env gene. A fragment of 444 bp was obtained and the sequence identity was found through BLAST ® application. The BLV prevalence for the department of Antioquia was 44 % (219/500). One of the PCR product was sequenced and classified as genotype 1; finding a 99 % identity with the sequence FJ808575.1. Samples from three regions of Antioquia department (East, North and Aburra Valley) were evaluated; the BLV prevalence was 70 %, 45 %, 31 % respectively. The molecular prevalence of BLV was between 16 and 88 % by the municipality. There was statistically significant difference (p<0.05) between the region of origin and the virus prevalence. The presence of BLV has been confirmed in the majority of the evaluated dairy units, which belong to one most important dairy regions in the department of Antioquia.